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Specialization of an Exonuclease III family enzyme in the repair of 3' DNA lesions during base excision repair in the human pathogen Neisseria meningitidis.

Silhan J, Nagorska K, Zhao Q, Jensen K, Freemont PS, Tang CM, Baldwin GS - Nucleic Acids Res. (2011)

Bottom Line: We now reveal further functional specialization at the level of 3'-PO(4) processing for NExo.However, no such functional redundancy exists for the 3'-phosphatase activity of NExo, and the cytotoxicity of 3'-blocking lesions is reflected in the marked sensitivity of a mutant lacking NExo to oxidative stress, compared to strains deficient in other base excision repair enzymes.A histidine residue within NExo that is responsible for its lack of AP endonuclease activity is also important for its 3'-phosphatase activity, demonstrating an evolutionary trade off in enzyme function at the single amino acid level.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK.

ABSTRACT
We have previously demonstrated that the two Exonuclease III (Xth) family members present within the obligate human pathogen Neisseria meningitidis, NApe and NExo, are important for survival under conditions of oxidative stress. Of these, only NApe possesses AP endonuclease activity, while the primary function of NExo remained unclear. We now reveal further functional specialization at the level of 3'-PO(4) processing for NExo. We demonstrate that the bi-functional meningococcal glycosylases Nth and MutM can perform strand incisions at abasic sites in addition to NApe. However, no such functional redundancy exists for the 3'-phosphatase activity of NExo, and the cytotoxicity of 3'-blocking lesions is reflected in the marked sensitivity of a mutant lacking NExo to oxidative stress, compared to strains deficient in other base excision repair enzymes. A histidine residue within NExo that is responsible for its lack of AP endonuclease activity is also important for its 3'-phosphatase activity, demonstrating an evolutionary trade off in enzyme function at the single amino acid level. This specialization of two Xth enzymes for the 3'-end processing and strand-incision reactions has not previously been observed and provides a new paradigm within the prokaryotic world for separation of these critical functions during base excision repair.

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Related in: MedlinePlus

NExo is an efficient 3′-phosphatase. (A) 50-PO4 (Table 1) was used as the substrate to measure removal of a 3′-blocking PO4 from an internal AP site. NExo (1 nM) was incubated with 50-PO4 (50 nM) under standard phosphatase assay conditions and aliquots removed at the indicated time points, quenched and resolved by denaturing 20% PAGE. (B) A number of reactions were performed with varying substrate concentrations using substrate 20-PO4 (Table 1); quantified data are shown with the best fit to the Michaelis–Menten equation with kcat = 0.49 ± 0.02/s and Km = 28 ± 6 nM.
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gkr905-F6: NExo is an efficient 3′-phosphatase. (A) 50-PO4 (Table 1) was used as the substrate to measure removal of a 3′-blocking PO4 from an internal AP site. NExo (1 nM) was incubated with 50-PO4 (50 nM) under standard phosphatase assay conditions and aliquots removed at the indicated time points, quenched and resolved by denaturing 20% PAGE. (B) A number of reactions were performed with varying substrate concentrations using substrate 20-PO4 (Table 1); quantified data are shown with the best fit to the Michaelis–Menten equation with kcat = 0.49 ± 0.02/s and Km = 28 ± 6 nM.

Mentions: The above data indicated that NExo is able to process 3′-PO4 lesions. We therefore characterized the kinetics of NExo against this specific lesion. To avoid enzyme pre-treatment synthetic 20-PO4 substrate (Table 1) was used for Michaelis–Menten kinetics (both 20-PO4 and 50-PO4 substrates were processed with same rate; data not shown). It was notable that NExo exhibited highly efficient 3′-phosphatase activity, with a kcat of 0.49/s (±0.02) and Km of 28 nM (±6) (Figure 6B). Therefore, NExo is a highly efficient phosphatase that recognizes and removes blocking 3′-PO4 lesion at internal gap sites and recessed 3′-termini.Figure 6.


Specialization of an Exonuclease III family enzyme in the repair of 3' DNA lesions during base excision repair in the human pathogen Neisseria meningitidis.

Silhan J, Nagorska K, Zhao Q, Jensen K, Freemont PS, Tang CM, Baldwin GS - Nucleic Acids Res. (2011)

NExo is an efficient 3′-phosphatase. (A) 50-PO4 (Table 1) was used as the substrate to measure removal of a 3′-blocking PO4 from an internal AP site. NExo (1 nM) was incubated with 50-PO4 (50 nM) under standard phosphatase assay conditions and aliquots removed at the indicated time points, quenched and resolved by denaturing 20% PAGE. (B) A number of reactions were performed with varying substrate concentrations using substrate 20-PO4 (Table 1); quantified data are shown with the best fit to the Michaelis–Menten equation with kcat = 0.49 ± 0.02/s and Km = 28 ± 6 nM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3300015&req=5

gkr905-F6: NExo is an efficient 3′-phosphatase. (A) 50-PO4 (Table 1) was used as the substrate to measure removal of a 3′-blocking PO4 from an internal AP site. NExo (1 nM) was incubated with 50-PO4 (50 nM) under standard phosphatase assay conditions and aliquots removed at the indicated time points, quenched and resolved by denaturing 20% PAGE. (B) A number of reactions were performed with varying substrate concentrations using substrate 20-PO4 (Table 1); quantified data are shown with the best fit to the Michaelis–Menten equation with kcat = 0.49 ± 0.02/s and Km = 28 ± 6 nM.
Mentions: The above data indicated that NExo is able to process 3′-PO4 lesions. We therefore characterized the kinetics of NExo against this specific lesion. To avoid enzyme pre-treatment synthetic 20-PO4 substrate (Table 1) was used for Michaelis–Menten kinetics (both 20-PO4 and 50-PO4 substrates were processed with same rate; data not shown). It was notable that NExo exhibited highly efficient 3′-phosphatase activity, with a kcat of 0.49/s (±0.02) and Km of 28 nM (±6) (Figure 6B). Therefore, NExo is a highly efficient phosphatase that recognizes and removes blocking 3′-PO4 lesion at internal gap sites and recessed 3′-termini.Figure 6.

Bottom Line: We now reveal further functional specialization at the level of 3'-PO(4) processing for NExo.However, no such functional redundancy exists for the 3'-phosphatase activity of NExo, and the cytotoxicity of 3'-blocking lesions is reflected in the marked sensitivity of a mutant lacking NExo to oxidative stress, compared to strains deficient in other base excision repair enzymes.A histidine residue within NExo that is responsible for its lack of AP endonuclease activity is also important for its 3'-phosphatase activity, demonstrating an evolutionary trade off in enzyme function at the single amino acid level.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK.

ABSTRACT
We have previously demonstrated that the two Exonuclease III (Xth) family members present within the obligate human pathogen Neisseria meningitidis, NApe and NExo, are important for survival under conditions of oxidative stress. Of these, only NApe possesses AP endonuclease activity, while the primary function of NExo remained unclear. We now reveal further functional specialization at the level of 3'-PO(4) processing for NExo. We demonstrate that the bi-functional meningococcal glycosylases Nth and MutM can perform strand incisions at abasic sites in addition to NApe. However, no such functional redundancy exists for the 3'-phosphatase activity of NExo, and the cytotoxicity of 3'-blocking lesions is reflected in the marked sensitivity of a mutant lacking NExo to oxidative stress, compared to strains deficient in other base excision repair enzymes. A histidine residue within NExo that is responsible for its lack of AP endonuclease activity is also important for its 3'-phosphatase activity, demonstrating an evolutionary trade off in enzyme function at the single amino acid level. This specialization of two Xth enzymes for the 3'-end processing and strand-incision reactions has not previously been observed and provides a new paradigm within the prokaryotic world for separation of these critical functions during base excision repair.

Show MeSH
Related in: MedlinePlus